O-GlcNAc transferase (OGT) is an essential mammalian enzyme that catalyzes a unique post-translational modification, O-GlcNAcylation. O-GlcNAcylation has been shown to modulate insulin signaling, gluconeogenesis, and other pathways connected to glucose metabolism. Hyper-O-GlcNAcylation leads to widespread transcriptional changes and has been linked to diabetic complications, cancer, and other pathologies. Therefore, it has been suggested that OGT could be a therapeutic target. A better understanding of the structure and function of OGT is critical for dissecting its biological roles, and small molecule inhibitors are widely sought by the scientific community. The goals of this proposal are i) to supply the structural basis for rational experiments to dissect the biochemical and cellular functions of OGT, ii) to provide validated small molecule inhibitors for use as cellular probes of OGT function, and iii) to develop a proteome-wide substrate profiling approach to identify OGT features required for substrate selection.